Project Summary There is an enormous need for the development of a new class of neurotransmitter (NT) sensors that are versatile, selective, sensitive and reliable to allow investigation of the neurobiological mechanisms of behavior and disease symptoms. This STTR proposal will focus on implementing a novel microarray probe for in vivo, real-time sensing of glutamate (GLU) and gamma-aminobutyric acid (GABA). GLU and GABA are NTs that are essential for normal brain function, neuronal activity, information processing and plasticity, and network synchronization. GLU is a major excitatory NT and GABA is a major inhibitory NT, and they must maintain a proper balance for the brain to operate normally. A GLU-GABA dysregulation plays a critical role in several brain disorders, including epilepsy (a disorder affecting 1.2% of Americans), dementia (a disorder that will affect 130 million worldwide by 2050) and Parkinson?s (a disorder affecting 1.5 million Americans today). A fundamental understanding of NT homeostasis would likely lead to new and effective therapeutic strategies for patients. Existing monitoring methods suffer from the inability to measure dynamics continuously, in real time. Currently, the primary method being used is microdialysis, which has very poor temporal resolution and is therefore not suitable to evaluate behavioral events that occur on a timescale of seconds or less. In contrast, biosensors are easy to miniaturize and are suitable for in vivo studies; they selectively oxidize GLU-GABA into a secondary electroactive product in the presence of enzymes, which is then detected by amperometry. Unfortunately, previously available biosensors have been cumbersome, have relied on externally applied reagents, and have been grossly inaccurate because their calibration has been carried out in vitro and not in vivo. We overcame these problems with GLU-GABA detection and demonstrated a biosensor array probe capable of a 26-fold higher sensitivity to GABA, a four-fold higher sensitivity to GLU, and no need for external reagents. Here, we propose to develop the next generation of our biosensor microelectrode array (MEA) probe technology that will allow GLU-GABA detection in real-time with the highest sensitivity, selectivity, and reliability. The probes will have the following new features: At least 2 GABA and 2 GLU sites for measuring concentrations at different positions within the brain. Also, it will feature a microfluidic channel for the introduction of chemicals in the immediate vicinity of the microelectrodes. Finally, we will incorporate an On-Demand In-situ Calibrator (ODIC) that runs in-situ sensor calibration for accurate detection. The specific aims of this project are: (i) microfabrication and characterization of platinum MEAs with ODIC on a silicon probe, (ii) surface modification and optimization of GLU-GABA probe in vitro and (iii) demonstrate real-time GLU-GABA detection in an epileptic rat brain. The proposed research would also enable alternative applications for the technology, including: point-of-use sensors for neurotoxins, reactive oxygen species, and disease biomarkers. The estimated market based on neurostimulation devices is $16 billion by 2024. If only 1% of that market is accessible by biosensor probe technology, it would still be sufficient justification for the proposed work.